One of the major biological actions of interferons (IFNs) is to deliver an antigrowth and/or antitumor effect in cells; a property that has allowed IFNs to be used clinically in the treatment of a variety of malignancies. The growth inhibitory effects of these cytokines are complex, and the induction of programmed cell death or "apoptosis" of tumor cells is variable and remains poorly understood. Expression of specific cellular genes induced by either IFN alpha/beta (Type 1) or IFN gamma (Type 2) is believed to mediate in part the biological actions of these cytokines. Gene transcription initiated by IFNs occurs primarily by the activation of the JAK/STAT signal transduction pathway. The Janus family of protein tyrosine kinases (JAK1, JAK2, JAK3 and TYK2) induces tyrosine phosphorylation and dimerization of specific transcription factors called STATs. These homodimeric or heterodimeric STAT complexes translocate to the nucleus where they bind to specific DNA elements found within the promoters of IFN regulated genes. In addition to IFN activation of the JAK/STAT pathway, other signaling pathways can be regulated as well by IFNs that in turn may contribute to the biological activities of these cytokines. The antigrowth but not the antiviral effects of type 1 IFNs in T cells have been described to require early signaling components of the T-cell receptor (TCR) signal transduction pathway: Lck, Zap70 and CD45, suggesting the existence of cross-talk between these two distinct signaling cascades. Recently, we characterized a variant of the Jurkat T-cell leukemic line that when stimulated with IFN alpha/beta undergoes apoptosis. Unlike the parental line where IFN alpha/beta induces an antigrowth effect without inducing apoptosis, the Jurkat variant exhibits a deficiency in mobilizing intracellular calcium and activation of the transcription factor NFAT, yet it displays intact activation of Zap70 and ERK1/2 kinases in response to TCR engagement. Preliminary results indicate that the defect in our Jurkat mutant must occur downstream of Zap-70 but upstream of PLC? kinase activation. Moreover, exposure of our Jurkat variant to multiple cycles of type I IFNs generated cells that became unresponsive to the apoptotic actions of IFNs. Careful evaluation of these clones revealed that these cells lacked expression of STAT2 or had a single amino acid substitution in a conserved motif not previously identified in the SH2 domain of STATs. These data suggest that in addition to STAT1; STAT2 is a critical component in the Jak/Stat pathway for the induction of apoptosis by type I IFNs. All together this raises an important question on how a defect in TCR signaling can lead to IFN ?/? induced apoptosis. Therefore it is important to elucidate the signaling mechanisms type I IFNs utilize to induce apoptosis and the crosstalk that might exist between TCR and IFN signaling pathways. Using these Jurkat cell lines as tools and model systems where type I IFNs clearly exert a distinct antigrowth response, will help us gain information regarding the molecular events and genes that must be induced or repressed by type I IFNs to turn on the "suicide" signal in tumor cells. It is the goal of our laboratory to elucidate the following:(i)The molecular mechanisms type I IFNs activate to drive apoptosis versus antiproliferative activity in tumor cells. Determine whether this is dependent on the induction of a specific set of IFN inducible genes that are only transcribed in apoptotic cells. Identify the mutation(s) harbored in the TCR signaling deficient Jurkat variant that renders them susceptible to undergo apoptosis in response to type I IFNs.(ii)Determine whether there are other TCR signaling molecules that are shared with type I IFN signal transduction pathway. (iii)Understand the role components of the JAK/STAT signaling cascade play in the apoptotic process initiated by type I IFNs. Study in depth the recently identified conserved motif in the SH2 domain of STATs and how this relates to the regulation of apoptosis induced by type I IFNs.